The goal of this project is to identify new genes for oral-facial clefts (OFC) by targeted capture and exome sequencing. Our study subjects are newly ascertained multiply affected Palestinian kindreds. Many recessively inherited phenotypes are common in this population as the result of consanguineous marriages. OFC genes discovered in the Palestinian population are informative for the American population, because for virtually every human trait, responsible genes discovered in any family, anywhere, also harbor mutations (usually different mutations) responsible for the trait in other families, worldwide. Since 1997, Palestinian families have cooperated with us in the discovery of genes responsible for severe traits. To identify genes for OFC, we will first complete enrollment of 200 multiply affected consanguineous Palestinian families. Second, we will evaluate all affected members of these families by targeted sequencing of all known syndromic and non- syndromic OFC genes, using an efficient approach that costs 25c per gene per sample. Third, to discover new OFC genes, we will exome sequence families not resolved by mutations in the known OFC genes. Finally, we will fully re-sequence each newly identified gene in DNA from a large series of cases and controls from the FaceBase BioRepository. Specifically, in AIM 1A, we will complete enrollment of ~864 affected individuals and a similar number of their informative unaffected relatives from 200 multiply affected Palestinian families, ~50% with non-syndromic OFC and ~50% with syndromic OFC. Thus far, we have consented, evaluated, and sampled 93 families in full and another 107 families in part. In AIM 1B, we will capture and sequence all known clefting genes in the ~864 affected individuals in these 200 families. As proof of principle, we developed a targeted panel with 137 known and candidate OFC genes and identified de novo damaging mutations in three children with syndromic clefting. The children's OFC phenotypes were consistent with their de novo genotypes. In AIM 2, families that do not harbor damaging alleles in known OFC genes will be exome sequenced at CIDR. In our lab, we will filter variants for rarity, for co-segregation with the phenotype in the family of origin, and for predicted effect on gene function. We will also use read depth-based algorithms to discover CNVs from exome data. Variants passing these filters, of any mutational type, from any gene, and at any exomic locale, will be reviewed and validated by Sanger sequencing, RT-PCR, and Q-PCR, as appropriate. As a preliminary study, we used exome sequencing to identify COL2A1 as the gene responsible for syndromic cleft palate in four multiply affected consanguineous Palestinian families. In AIM 3 we will re-sequence candidate genes from Aim 2 in 1500 unrelated subjects with OFCs and 2500 controls available through FaceBase. Our goal is to determine the variant spectrum of each gene and to identify any genes with a more severe mutational burden in cases than in controls. Gene discovery in these uniquely informative, well-characterized kindreds with OFCs offers the opportunity to identify new genes for this common birth defect.